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Int. J. Biol. Sci. 2012, 8
Ivyspring
International Publisher
International Journal of Biological Sciences 2012; 8(4):533-547. doi: 10.7150/ijbs.3753
Research Paper
Isoalantolactone Induces Reactive Oxygen Species Mediated Apoptosis in Pancreatic Carcinoma PANC-1 Cells Muhammad Khan1,2, Chuan Ding2, Azhar Rasul1, Fei Yi1, Ting Li1, Hongwen Gao1, Rong Gao1, Lili Zhong1, Kun Zhang1, Xuedong Fang1 and Tonghui Ma1,2 1. Central Research Laboratory, Jilin University Bethune Second Hospital, Changchun 130041, P. R. China; 2. Membrane Channel Research Laboratory, Northeast Normal University, Changchun 130024, P. R. China. Corresponding author: Tonghui Ma, Central Research Laboratory, Jilin University Bethune Second Hospital, Changchun 130041, P. R. China. Tel: +86-431-88796667. Fax: +86-431-88796667. E-mail:
[email protected] © Ivyspring International Publisher. This is an open-access article distributed under the terms of the Creative Commons License (http://creativecommons.org/ licenses/by-nc-nd/3.0/). Reproduction is permitted for personal, noncommercial use, provided that the article is in whole, unmodified, and properly cited.
Received: 2011.11.05; Accepted: 2012.03.22; Published: 2012.03.28
Abstract Isoalantolactone, a sesquiterpene lactone compound possesses antifungal, antibacteria, antihelminthic and antiproliferative activities. In the present study, we found that isoalantolactone inhibits growth and induces apoptosis in pancreatic cancer cells. Further mechanistic studies revealed that induction of apoptosis is associated with increased generation of reactive oxygen species, cardiolipin oxidation, reduced mitochondrial membrane potential, release of cytochrome c and cell cycle arrest at S phase. N-Acetyl Cysteine (NAC), a specific ROS inhibitor restored cell viability and completely blocked isoalantolactone-mediated apoptosis in PANC-1 cells indicating that ROS are involved in isoalantolactone-mediated apoptosis. Western blot study showed that isoalantolactone increased the expression of phosphorylated p38 MAPK, Bax, and cleaved caspase-3 and decreased the expression of Bcl-2 in a dose-dependent manner. No change in expression of phosphorylated p38 MAPK and Bax was found when cells were treated with isoalantolactone in the presence of NAC, indicating that activation of these proteins is directly dependent on ROS generation. The present study provides evidence for the first time that isoalantolactone induces ROS-dependent apoptosis through intrinsic pathway. Furthermore, our in vivo toxicity study demonstrated that isoalantolactone did not induce any acute or chronic toxicity in liver and kidneys of CD1 mice at dose of 100 mg/kg body weight. Therefore, isoalantolactone may be a safe chemotherapeutic candidate for the treatment of human pancreatic carcinoma. Key words: Isoalantolactone; PANC-1; ROS; Apoptosis; NAC.
Introduction Pancreatic cancer is the fourth most common cause of cancer-related deaths in United States [1]. Despite advancements in diagnosis, surgery, radiotherapy and chemotherapy, the overall 5 year survival rate remains less than 3 years while the median survival rate is about 6 months [2]. Surgical abscission remains the only option for long term survival of patients. However, difficulty in achieving early diagnosis and aggressive nature of this type of cancer limit
the surgical operation to only about 10% patients. Therefore, the majority of pancreatic cancer patients are treated with chemotherapy [3]. Currently, gemcitabine (Gem) is the most effective chemotherapeutic drug for pancreatic cancer. However, even with this drug, the overall survival rate remains considerably low. Moreover, Gem is highly toxic to tumor cells as well as normal cells [4, 5]. Several other cytotoxic and chemotherapy agents such as cisplatin, fluorouracil, http://www.biolsci.org
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Int. J. Biol. Sci. 2012, 8 docetaxel and irinotecan have been tested as single agent or in combination with gemcitabine for pancreatic cancer, however most of these studies failed to ameliorate overall patient survival compared to gemcitabine [3]. Furthermore, most of these drugs have been shown to cause severe hepatotoxicity [6]. As pancreatic cancer responds poorly to the existing conventional chemotherapy, therefore it is important to study the anti-pancreatic cancer mechanism using newly identified effective anticancer compounds that exhibit low toxicity on normal cells. A number of recent reports indicated that phytochemicals targeting ROS metabolism can selectively kill the cancer cells by raising the level of ROS above a toxic threshold. As cancer cells contain higher level of endogenous ROS than normal cells, the toxic threshold can be easily achieved in cancer cells compared to normal cells [7, 8]. In the present study, we performed high throughput screening of compound library from Chinese herbs, using pancreatic cancer cell line PANC-1, in the presence or absence of NAC, a specific ROS inhibitor. This screening method allowed us to identify anti-pancreatic cancer compounds targeting ROS metabolism. Isoalantolactone, a sesquiterpene lactone compound was identified as a potent inhibitor of pancreatic carcinoma cells during screening process. Sesquiterpene lactones are plant-derived compounds used in the manufacture of traditional medicines for the treatment of inflammatory diseases, headache and infections [9]. Over the past few years, a large body of pharmacological studies has provided convincing evidences of the anticancer property of sesquiterpene lactones against various human cancer cell lines [10, 11]. At present, several sesquiterpene lactone compounds are in cancer clinical trials against breast, colorectal, kidney, prostate, acute myeloid leukemia, acute lymphoblastic leukemia, and non small lung cancer treatment [10]. However, the anti-pancreatic cancer potential of sesquiterpene lactone compounds is not well studied. Isoalantolactone is one of the major sesquiterpene lactone compounds, isolated from the roots of Anula helenium and possesses multiple biological activities including antibacteria, antifungal, antihelminthic and antiproliferative [12]. The above findings encouraged us to study the anti-pancreatic cancer potential of isoalantolactone as well as its toxicity on normal cells in vitro and hepatotoxicity and nephrotoxicity in vivo.
Materials and Methods Reagents
Tauto Biotech Co., Ltd. (Shanghai, China) and purity (> 99%) was determined by HPLC. RNase A, Propidium iodide (PI), calcein acetoxymethylester (Calcein AM), Hoechst 33258, 10-Nnonyl Acridine Orange (NAO), Dimethyl Sulfoxide (DMSO), [3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide] (MTT), Dulbecco's Modified Eagle's Medium (DMEM), DMEM/F12 medium, fatal bovine serum (FBS), penicillin and streptomycin were purchased from Sigma (Beijing, China). Apoptosis assay kit, Reactive oxygen species kit, JC-1, and Antibodies specific to p-p38 MAPK, Bax, Bcl-2, cytochrome c, and caspase-3 were purchased from Beyotime institute of Technology (Shanghai, China). Antibodies specific to β-actin and horseradish peroxidase-conjugated secondary antibodies (goat-anti-rabbit, goat-anti-mouse) were purchased from Santa Cruz (Beijing, China).
Cell Culture and Treatments The human pancreatic carcinoma PANC-1, BxPC3, HPAC cells and monkey non-tumorigenic COS-7 cells were obtained from Shanghai Cell Bank. PANC-1, BxPC3 and COS-7 cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fatal bovine serum (FBS), 100 units/mL penicillin and 100 µg/mL streptomycin whereas HPAC cells were cultured in DMEM/F12 medium supplemented with 10% (FBS), 100 units/mL penicillin and 100 µg/mL streptomycin and maintained at 37°C with 5% CO2 in humidified atmosphere. Cells were treated with various concentrations of isoalantolactone dissolved in DMSO with a final DMSO concentration of 1% for 24 h. DMSO treated cells were used as control.
Determination of Cell Viability Cell viability was assessed by MTT assay as described previously [13]. Briefly PANC-1, BxPC3, and HPAC cells were treated with dimethyl sulfoxide (DMSO) or isoalantolactone in the presence or absence of 3 mM NAC for 24 h. Following treatment, the MTT reagent was added (500µg/mL) and cells were further incubated at 37°C for 4 h. Subsequently 150 μL DMSO was added to dissolve farmazan crystals and absorbance was measured at 570 nm in a microplate reader (Thermo Scientific). The percentage of cell viability was calculated as follows: Cell viability (%) = (A570sample – A570blank) / (A570control – A570blank)×100 The IC50 Values were calculated using GraphPad Prism 5.
Isoalantolactone (IALT) was purchased from
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Int. J. Biol. Sci. 2012, 8 Live/Dead assay Live and dead cells were quantified using the fluorescent probes calcein acetoxymethylester (calcein AM) and propidium iodide (PI). Celcein AM is cell membrane permeable and stains only viable cells whereas PI is cell membrane impermeable and stains only dead cells. To determine the effect of isoalantolactone on cancer cells and normal cells, pancreatic carcinoma cells (PANC-1) and normal cells (COS-7) were treated with 20 and 40 µM isoalantolactone in the presence or absence of NAC for 24 h. Subsequently, treated and untreated cells were collected, washed with phosphate buffered saline (PBS) and incubated with PBS solution containing 2 µM calcein AM and 4 µM PI in the dark for 20 min at room temperature. After washing, cells were resuspended in PBS and analyzed for the fluorescence of calcein and PI by flow cytometry (Beckman Coulter, Epics XL).
Apoptosis assay by Annexin V-FITC and propidium iodide (PI) staining PANC-1 cells were treated with isoalantolactone in a dose- and time-dependent manner in the presence or absence of 3 mM NAC. After treatment, cells were harvested, washed with PBS, and resuspended in 200 µL of binding buffer containing 5 µL Annexin V and put in the dark for 10 min according to the kit instructions (Beyotime, Shanghai, China). After incubation, cells were labeled with 10 µL PI and samples were immediately analyzed by flow cytometry (Beckman Coulter, Epics XL).
Hoechst 33258 staining for Nuclei Condensation and Fragmentation PANC-1 cells were treated with 40 µM isoalantolactone for various time points (0, 4, 12, and 24 h). The cells were fixed with 4% paraformaldehyde for 30 min at room temperature. After washing with PBS, cells were stained with Hoechst 33258 (50 µg/mL) at 37°C for 20 min in the dark. At the end, the cells were washed and resuspended in PBS for the observation of nuclear morphology under fluorescence microscope (Olympus 1x71).
Cell cycle analysis PANC-1 cells were treated with 20 and 40 µM isoalantolactone for 24 h. After treatment, cells were harvested, washed with PBS and fixed with 70% ethanol at 4°C for overnight. After washing twice with PBS, cells were stained with a solution containing 50 μg/mL PI and 100 μg/mL RNase A for 30 min in the dark at room temperature. The DNA contents for cell cycle phase distribution were analyzed by flow cy-
tometry (Beckman Coulter, Epics XL) using Cell Quest software.
Measurement of Reactive Oxygen Species (ROS) The intracellular changes in ROS generation were measured by staining the cells with 2', 7' -dichlorofluorescein-diacetate (DCFH-DA) as described previously [13]. The fluorescent dye DCFH-DA is a cell membrane permeable and is converted into cell membrane impermeable nonfluorescent compound DCFH by intracellular esterases. Oxidation of DCFH by reactive oxygen species produces a highly fluorescent DCF. The fluorescence intensity of DCF inside the cells is proportional to the amount of peroxide produced. Briefly PANC-1 cells were treated with 20 and 40 µM isolantolactone for 24 h. After treatment, cells were further incubated with 10 μM DCFH-DA at 37°C for 30 min. Subsequently, cells were harvested, rinsed, re-suspended in PBS, filtered with 300 apertures and analyzed for 2', 7' -dichlorofluorescein (DCF) fluorescence by flow cytometry.
Measurement of Mitochondrial Membrane Lipid Peroxidation Mitochondrial membrane lipid peroxidation was determined by measuring the oxidation of intracellular cardiolipin by staining the cells with 10-N-nonyl acridine orange (NAO), a probe specific for mitochondrial membrane cardiolipin [14]. Briefly PANC-1 cells were treated with 20 and 40 µM isoalantolactone for 24 h. After washing with PBS, cells were further incubated with 5 µM NAO in the dark at room temperature for 30 min. After washing, samples were analyzed by flow cytometry.
Measurement of Mitochondrial Membrane Potential (MMP) MMP was determined using JC-1 probe (Beyotime) as described previously [15, 16]. JC-1 is widely used to monitor mitochondrial membrane depolarization. In healthy cells with high mitochondrial membrane potential, JC-1 spontaneously forms complexes known as J-aggregates with intense red fluorescence. Whereas, in apoptotic or unhealthy cells with low mitochondrial membrane potential, JC-1 remains in the monomeric form, which shows only green fluorescence. Thus mitochondrial depolarization is indicated by a decrease in red/green fluorescence intensity ratio. Briefly after treating the cells with 20 and 40 µM isoalantolactone for 24 h, cells were stained with 10 µM of JC-1 for 25 min at 37°C. After
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Int. J. Biol. Sci. 2012, 8 washing, cells were analyzed for the decrease in red/green fluorescence by flow cytometry.
Immunobloting Proteins were isolated from control and isoalantolactone-treated cells as described previously [13]. 40 µg proteins were electrophoresed on 10% SDS-PAGE and transferred to PVDF membrane. After blocking with 5% (w/v) non-fat milk and washing with Tris-buffered saline-Tween solution (TBST), membranes were incubated overnight at 4°C with p-p38 MAPK (1:1000), Bcl-2 (1:1000), Bax (1:300), cytochrome c (1:200), caspase-3 (1:500) and β-actin (1:400) antibodies respectively. After washing, the blots were incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG or goat anti-mouse IgG secondary antibodies (1:5000) for 1 h at room temperature. After washing with TBST, signals were detected using ECL plus chemiluminescence kit on X-ray film (Millipore Corporation). All the bands obtained were quantified by densitometry using Image J software.
In Vivo Studies In vivo studies for acute and chronic toxicity were conducted on 10-12 week old CD1 mice weighing 27-30 g. The mice were maintained in a specific pathogen-free grade animal facility on a 12-h light/dark cycles at 25±2°C. Mouse procedures were approved by the Experimental Animal Committee of Jilin University. Mice were randomly divided into four groups. Group A (n = 4) administered with 50 µL DMSO intraperitonially for 7 days; Group B (n = 4) administered with isoalantolactone (100 mg/kg body weight) in 50 µL DMSO intraperitonially for 7 days; Group C (n = 4) administered with 50 µL DMSO for 30 days and Group D (n = 4) administered with isoalantolactone (100 mg/kg body weight) in 50 µL DMSO intraperitonially for 30 days. At the first and last day of the experiments, the body weight of each mouse was measured. At the end of experiments (at dose day 7 for acute toxicity & dose day 30 for chronic toxicity), mice were anesthetized using Pentobarbital sodium (50 mg/kg ip), blood was collected via cardiac puncture, allowed to clot for 10 min and centrifuge at 1000×g for 10 min at room temperature. Serum was separated and stored at -20°C until analysis. The liver and kidneys were excised and processed for hematoxylin and eosin staining followed established procedures.
Serum Biomarker Analysis The acute and chronic toxicity of isoalantolactone on liver and kidneys was determined by meas-
uring the serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), total bilirubin (TBIL), blood urea nitrogen (BUN) and creatinine (Cr) using blood auto analyzer (Hitechi 7170).
Statistical Analysis The results are expressed as Mean ± SEM and statistically compared with control group or within the groups using one way ANOVA followed by Tukey’s Multiple Comparison Test. Student’s T-test was used to determine significance when only two groups were compared and p
